Patent application title: BLUEBERRY RED RINGSPOT VIRUS, SEQUENCES, PROMOTERS, AND USES THEREOF
Richard F. Allison (Leslie, MI, US)
Jerri Gillett (East Lansing, MI, US)
Christy Mecey (Leslie, MI, US)
Board of Trustees of Michigan State of University
IPC8 Class: AC07H2104FI
Class name: Dna or rna fragments or modified forms thereof (e.g., genes, etc.) probes for detection of specific nucleotide sequences or primers for the synthesis of dna or rna probes for detection of microbial nucleotide sequences
Publication date: 2009-01-22
Patent application number: 20090023909
A nucleic acid sequence of the blueberry red ringspot virus is disclosed.
Also disclosed are putative promoter regions of the sequence and promoter
regions capable of directing transgene expression in plants, including
tissue-specific expression. Also disclosed are expression vectors,
transformed plant cells and plants containing a blueberry red ringspot
virus promoter and an encoded product for expression. Methods for
diagnosis of blueberry red ringspot virus infection are also provided.
6. A nucleic acid probe or primer of at least 10 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 1 or the complement thereof, wherein said probe or primer specifically hybridizes under stringent conditions to a blueberry red ringspot virus nucleic acid sequence.
7. The nucleic acid probe or primer of claim 6 which specifically hybridizes under stringent conditions to a promoter region comprising BRRV promoter region A as set forth in SEQ ID NO: 2, BRRV promoter region B as set forth in SEQ ID NO: 3, BRRV promoter region C as set forth in SEQ ID NO: 4, BRRV promoter region D as set forth in SEQ ID NO: 5, BRRV promoter region E as set forth in SEQ ID NO: 6, BRRV promoter region F as set forth in SEQ ID NO: 7, BRRV promoter region G as set forth in SEQ ID NO: 8, or a BRRV promoter region as set forth in SEQ ID NO: 9.
8. A recombinant nucleic acid comprising a promoter from the blueberry red ringspot virus operably linked to a DNA sequence encoding a polypeptide or an RNA.
9. The recombinant nucleic acid of claim 8 wherein the promoter comprises BRRV promoter region A as set forth in SEQ ID NO: 2, BRRV promoter region B set forth in SEQ ID NO: 3, BRRV promoter region C set forth in SEQ ID NO: 4, BRRV promoter region D set forth in SEQ ID NO: 5, BRRV promoter region E set forth in SEQ ID NO: 6, BRRV promoter region F set forth in SEQ ID NO: 7, BRRV promoter region G as set forth in SEQ ID NO: 8, or a BRRV promoter region as set forth in SEQ ID NO: 9.
10. The recombinant nucleic acid of claim 9 wherein the promoter region is operably linked to a gene encoding a polypeptide or an RNA.
11. A transgenic plant cell comprising the recombinant nucleic acid of claim 10.
12. A transgenic dicotyledonous plant comprising the plant cell of claim 11.
13. A transgenic monocotyledonous plant comprising the plant cell of claim 11.
14. The transgenic plant of claim 11, wherein the plant is of the species Arabidopsis thaliana.
15. A method for transforming a plant cell comprising transforming a plant cell with a recombinant DNA construct comprising a blueberry red ringspot virus promoter and a DNA sequence which encodes a polypeptide or an RNA; wherein the promoter regulates the transcription of the DNA sequence.
16. A method for diagnosing the presence of blueberry red ringspot virus in a host plant comprising (a) incubating a nucleic acid sample obtained from a plant suspected of containing the virus with a nucleic acid probe of claim 6 under conditions in which the probe can hybridize with any blueberry red ringspot virus nucleic acid present in the sample and (b) detecting the presence of any hybridization complex formed.
17. A method for diagnosing the presence of blueberry red ringspot virus in a host plant comprising (a) providing two oligonucleotides which are primers for a polymerase chain reaction method and which flank a target sequence which lies within a blueberry red ringspot virus nucleic acid as shown in FIG. 2 or the complement thereof; (b) incubating the oligonucleotides with a nucleic acid sample obtained from a plant suspected of containing the virus; (c) amplifying the target DNA sequence if it is present in the nucleic acid sample by the polymerase chain reaction method and (d) detecting the presence of the any amplified target DNA sequence.
18. A recombinant nucleic acid comprising a sequence having promoter activity in a plant cell and which hybridizes under high stringency conditions to a fragment of a blueberry red ringspot virus nucleic acid which has promoter activity in the plant cell, or the complement thereof.
19. The recombinant nucleic acid of claim 18 comprising BRRV promoter region A as set forth in SEQ ID NO: 2, BRRV promoter region B as set forth in SEQ ID NO: 3, BRRV promoter region C as set forth in SEQ ID NO: 4, BRRV promoter region D as set forth in SEQ ID NO: 5, BRRV promoter region E as set forth in SEQ ID NO: 6, or BRRV promoter region F as set forth in SEQ ID NO: 7.
20. The recombinant nucleic acid of claim 19 wherein the plant cell is a dicotyledonous plant cell.
21. The recombinant nucleic acid of claim 19 wherein the plant cell is a monocotyledonous plant cell.
22. The recombinant nucleic acid of claim 18 wherein the sequence comprises a consensus promoter sequence as set forth in SEQ ID NO: 10.
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 10/793,454, filed Mar. 4, 2004, which is a continuation of International Application No. PCT/US02/28260, filed Sep. 5, 2002, which claims the benefit of U.S. Provisional Application Ser. No. 60/318,050, filed Sep. 7, 2001, all of which are hereby incorporated by reference herein.
BACKGROUND OF THE INVENTION
(1) Field of the Invention
This invention relates to nucleic acid sequences of the blueberry red ringspot virus and, more particularly, to the nucleic acid sequence of the virus, the identification of promoters, and the use of the promoters in the expression of recombinant genes in transgenic plants, including tissue-specific expression in plants. The invention also relates to sequences of the blueberry red ringspot virus useful in the diagnosis of disease in plants, and related methods thereof.
(2) Description of the Related Art
Recombinant viral promoters can be used to direct the expression of operably linked heterologous genes. Such expression can occur in a transgenic environment. For expression of transgenes in plants, a promoter from Cauliflower Mosaic Virus (CaMV) is widely used, for example as disclosed in U.S. Pat. No. 6,255,560 to Fraley et al. Expression of transgenes in plants under the control of a CaMV promoter tends to be at high levels and show little tissue- or cell-type specificity. This virus is unusual, in that it appears to comprise only two promoters. One promoter appears to control the transcription of the entire viral genome into an RNA copy. This promoter (the "35S" promoter) is a tandem repeat of an approximately 350 base pair sequence. Within this sequence are domains involved with tissue specific expression of genes expressed from the promoter (Odell, J. T., Nagy, F. and Chua, N. H. (1985) Identification of DNA sequences required for the activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810-812; Benfey, P. N. and Chua, N. H. (1990) The Cauliflower Mosaic Virus 35S promoter: combinatorial regulation of transcription in plants. Science 250: 959-966; Daubert, S. D., et al., (1984) Expression of Disease Symptoms in CaMV Genomic Hybrids. Journal of Molecular and Applied Genetics 202: 1043-1045; Dixon, L. K., et al., (1983) Mutagenesis of Cauliflower Mosaic Virus. Gene 25: 189-199; Mesnard, J., et al. (1990) The Cauliflower Mosaic Virus Gene III product is a non-sequence specific DNA binding protein. Virology 174: 622-624; Pfeiffer, P. and Hohn, T. (1983) Involvement of reverse transcriptase in the replication of cauliflower mosaic virus. Cell 33: 781-789; Takatsuji, H., et al. (1992) Cauliflower Mosaic Virus reverse transcriptase--activation by proteolytic processing and functional alteration by terminal deletion. Journal of Biological Chemistry 267: 11579-11585; Thomas, C. L. et al. (1992) A mutation in Cauliflower Mosaic Virus Gene I interferes with virus movement but not with virus replication. Virology 74: 1141-1148). This promoter has proven useful for directing the expression of heterologous genes in transgenic plants.
Because transgenes under CaMV promoter control may not be suitable for all uses, there is a need for the identification and characterization of more plant virus promoters. For example, there is the need for tissue specific promoters that can direct expression of an operably linked gene to a subset of tissues within a transgenic plant. Furthermore, there is a need for a strategy for identifying putative promoters, and a further need to demonstrate the operability of a putative promoter in a transgenic environment.
The blueberry red ringspot virus is a virus with a limited host range: it is believed to infect only blueberry plants. Disease symptoms are observed primarily in the months of July, August and September, and comprise red spots primarily on upper leaf surfaces (Hutchinson, M. T. (1950) Can you recognize the symptoms of stunt disease? Proceedings 19th Annual Blueberry Open House 19: 9-11; Ramsdell, D. C., Kim, K. S. and Fulton, J. P. (1987) Red Ringspot of Blueberry. In: Converse, R. H. (ed.) Virus Diseases of Small Fruits. U.S. Department of Agriculture, Agr. Res. Svc., Washington, D.C. Handbook 631; Kim, K. S., Ramsdell, D. C., Gillett, J. M. and Fulton, J. P. (1981) Virions and substructural changes associated with blueberry red ringspot disease. Phytopathology 71: 673-678; Gillett, J. M. (1988) Physical and Chemical properties of Blueberry Red Ringspot Virus. Master's thesis, Michigan State University; Hutchinson, M. T. and Varney, E. H. (1954) Ringspot: A virus disease of cultivated blueberry. Plant Disease Reports 38: 260-262). A sequence of a putative blueberry red ringspot virus has been published on the http World Wide Web at ncbi.nlm.nih.gov with accession numbers NC 003138 and AF404509. This sequence has a length of 8,303 base pairs. No promoters are disclosed or identified in these sequence listings. Furthermore, this sequence has several differences with the sequence of the virus of the present invention as disclosed herein.
BRIEF DESCRIPTION OF THE INVENTION
The present invention involves the discovery and characterization of the nucleic acid sequence of the genome of blueberry red ringspot virus (BRRV). The genome comprises a circular, double-stranded DNA molecule. The molecule has a length of at least 8,241 base pairs. Promoters are identified in the sequence based upon analysis of sequence structure. Recombinant promoters derived from BRRV DNA are used to direct gene expression.
In some embodiments, the present invention is directed to a fragment of the viral nucleic acid which putatively functions as a promoter in plant cells. Each of these putative promoter regions comprises a "TATATAA box" or a "TATA box" and a nearby open reading frame located 3' to the TATATAA box or TATA box. In another embodiment, a consensus sequence for the blueberry red ringspot putative promoters is provided. Also within the scope of the invention are methods for identifying putative promoter sequences of the Blueberry Red Ringspot Virus.
In preferred embodiments, the invention is drawn to a recombinant nucleic acid comprising a fragment of the full-length nucleic acid of the BRRV which functions as a promoter (a "BRRV promoter"). In a preferred embodiment, the present invention provides a recombinant nucleic acid comprising a BRRV promoter operably linked to a gene for expression as a transgene in a host organism or cell. The transgene preferably encodes a polypeptide or a regulatory RNA molecule. Preferably, the host organism is a plant, and the host cell is a plant cell. In another preferred embodiment, a recombinant nucleic acid comprising a BRRV promoter operably linked to a gene for expression as a transgene provides tissue-specific expression of the transgene in a transformed host plant or a descendant thereof. Preferably, the tissue-specific expression is directed to root tissue or to leaf tissue. More preferably, the tissue-specific expression is directed to root tissue.
In some embodiments, the invention is drawn to a transgenic plant or a transgenic plant cell comprising a recombinant BRRV promoter operably linked to a transgene. In some embodiments, promoter modifications, for example deletions and tandem duplications, provide alternative promoter constructs providing altered transcription patterns. Altered transcription patterns comprise alterations in quantity, timing, and/or tissue specificity in the expression of an operably linked transgene.
The present invention is directed, in some embodiments, to the nucleic acid sequence of the blueberry red ringspot virus and to fragments thereof comprising at least 10 consecutive nucleotides of the full-length sequence. Such fragments provide probes and primers to detect blueberry red ringspot virus DNA in diagnostic tests to ascertain whether a susceptible host plant is infected with the virus.
In some embodiments, the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence capable of hybridizing under stringent conditions to a nucleic acid comprising a sequence of the blueberry red ringspot virus, or a complement thereof.
Among the several advantages achieved by the present invention, therefore, may be noted: the nucleic acid sequence of the blueberry red ringspot virus genome; putative promoters of the blueberry red ringspot virus; a consensus sequence of blueberry red ringspot virus promoters; a vector comprising a recombinant BRRV promoter operably linked to a sequence encoding a polypeptide or an RNA; a transgenic plant or transgenic plant cell comprising a recombinant BRRV promoter operably linked to a sequence encoding a polypeptide or an RNA; a recombinant BRRV promoter that can direct expression of an operably linked DNA sequence encoding a polypeptide or an RNA; a recombinant BRRV promoter that can direct tissue-specific expression of an operably linked DNA sequence encoding a polypeptide or an RNA; a method for transforming a plant or a plant cell with a DNA molecule comprising a recombinant BRRV promoter and an operably linked DNA sequence encoding a polypeptide or an RNA; and a diagnostic method for detecting the presence of blueberry red ringspot virus in a host plant.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 represents a map (not drawn to scale) of the blueberry red ringspot virus genome.
FIG. 2 represents a comparative alignment of blueberry red ringspot virus putative promoter sequences `a` (nts 221-373 of SEQ ID NO:2) in row one, `d` (nts 235-384 of SEQ ID NO:5) in row two, `e` (nts 131-329 of SEQ ID NO:6) in row three, `f` (nts 205-354 of SEQ ID NO:7) in row four, `g` (nts 257-408 of SEQ ID NO:8) in row five, `b` (nts 176-324 of SEQ ID NO:3) in row six, and `c` (nts 240-389 of SEQ ID NO:4) in row seven, and a consensus DNA sequence for a putative blueberry red ringspot virus promoter derived from the sequence data (SEQ ID NO:10) in row eight.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, blueberry red ringspot virus, the pathogen responsible for causing red ringspot disease in blueberry plants, has been sequenced and characterized by standard methods, such as those disclosed in Sambrook, J. C., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. The virus comprises a circular genome of double stranded DNA. A linearized map of the circular genome (and its putative promoters and open reading frames, discussed below) is shown in FIG. 1, and the sequence in linearized form (SEQ ID NO: 1) is shown in FIG. 2. The virus is sequenced by subcloning restriction fragments into sequencing vectors. Fragments are generated using more than one restriction enzyme, and common sequences are used to order the sequence fragments. Because isolates of the virus from different individual infected plants provide slightly different sequences, a full-length sequence of a BRRV DNA can be different from the sequence disclosed herein. The BRRV sequence comprises at least 8214 base pairs. The nucleic acid sequence of the virus in the present invention (SEQ ID NO: 1) represents a composite assembled from sequences from several isolates and comprises most of the BRRV sequence.
Fragments of the full-length sequence are used as probes and/or as PCR primers to detect the presence of the virus in susceptible host plants according to methods known in the art. Such fragments can be, preferably, at least 10 consecutive nucleotides in length, at least 15 consecutive nucleotides in length, at least 20 consecutive nucleotides in length, at least 50 consecutive nucleotides in length, at least 100 consecutive nucleotides in length or greater up to the full-length viral sequence. Such fragments or complements thereof specifically hybridize to the blueberry red ringspot DNA sequence as shown in FIG. 2, or the complement thereof. Specific hybridization results in hybridization of the fragments or complements thereof to the blueberry red ringspot virus or to a target sequence thereof, preferentially over other DNA molecules that might be present in a sample tested. Hybridization conditions for obtaining such specificity, preferably, involve high stringency conditions as are well known in the art (see Sambrook et al, supra).
Diagnostic methods based upon the probes and/or primers are also within the scope of the present invention. In one diagnostic approach, a nucleic acid sample obtained from a plant suspected of containing the virus is incubated with a nucleic acid probe of the present invention. Incubation conditions are such that the probe will specifically hybridize with any blueberry red ringspot virus nucleic acid that might be present in the sample. Any resultant hybridization complexes formed are then detected using methods well known in the art.
PCR methods are also contemplated in diagnostic methods in which, preferably, two oligonucleotide primers of the present invention serve as primers for a polymerase chain reaction method. Primers are selected from the sequence as shown in FIG. 2 or the complement thereof, to flank a target sequence which lies within a blueberry red ringspot virus nucleic acid. Any target DNA that might be in a nucleic acid sample obtained from a plant suspected of containing the virus is then amplified by standard methods using the primers. Criteria for selecting primer sequences are well known in the art. The presence of an amplified target DNA sequence is then detected by methods known in the art.
In some embodiments, the present invention includes methods of identifying putative promoters of the blueberry red ringspot virus. In this method, BRRV sequences with the properties of "TATATAA boxes" are identified using computer and/or manual methods (see, e.g., Boyer, T. G., and Maquat, L. E. (1990). Minimal sequence and factor requirements for the initiation of transcription from an atypical TATATAA box-containing housekeeping promoter. J. Biol. Chem. 265: 20524-20532). In preferred embodiments, sequence analysis is conducted with the aid of a digital computer programmed with algorithms known in the art, preferably a GCG (University of Wisconsin-Madison) "FIND" software program. In this method, TATATAA-box elements within the BRRV sequence are examined for ATG translational start sites and for classical cis-acting elements, such as AS-1/AS-2-like motifs and GATA boxes. The presence in a sequence of at least one TATATAA-box, at least one classical cis-acting element, and at least one translation start site located 100 base pairs or less downstream from the TATATAA-box provide criteria for designating a BRRV sequence as a putative BRRV promoter.
Analysis of the sequence of the blueberry red ringspot virus by the above method reveals the presence of putative promoters and open reading frames. Seven putative promoters identified using the above criteria are designated "BRRV Promoter A" (SEQ ID NO: 2), "BRRV Promoter B" (SEQ ID NO: 3), "BRRV Promoter C", (SEQ ID NO: 4) "BRRV Promoter D" (SEQ ID NO: 5), "BRRV Promoter E" (SEQ ID NO: 6), "BRRV Promoter F" (SEQ ID NO: 7), and "BRRV Promoter G. (SEQ ID NO: 8)" Their positions in the blueberry red ringspot virus genome are determined by referring to the map in FIG. 1 or the BRRV sequence (SEQ ID NO: 1). In addition, FIG. 1 indicates the relative positions of known open reading frames with respect to the putative promoters. The presence of 7 putative promoters is an unexpected result; another virus within the same family (Caulimoviridae), the cauliflower mosaic virus, is known to comprise only 2 promoters, including at least one which transcribes the entire genome. It is believed that the promoters of the present invention can function as monocotyledonous and dicotyledonous plant cell promoters. However, functional assays of recombinant putative promoter (see below) have revealed promoter activity from only two BRRV promoters.
Open reading frame (ORF) regions are identified as the portion of the sequence extending after the TATA-box element from a start codon to a stop codon. ORF regions are illustrated in FIGS. 1 and 2.
In some embodiments, the promoters of the present invention are incorporated into vectors that can replicate in a host organism. Such vectors are independently-replicating nucleic acid molecules including, for example, plasmids, phages, and viruses. The vectors can be used to transform eukaryotic cells, for example, plant cells, fungi or other eukaryotic cells. Such transformed plant cells include monocotyledonous and dicotyledonous plant cells.
In some embodiments, the invention is a nucleic acid comprising the promoter region operably linked to a sequence encoding a polypeptide or an RNA molecule. Non-limiting examples of a polypeptide include, a fluorescent protein (See, e.g., Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994), Green fluorescent protein as a marker for gene expression. Science 263: 802-805); chloramphenicol acetyl transferase; a protein of pharmaceutical interest; and a protein providing a host plant with pest resistance. A non-limiting example of an RNA molecule that can be expressed from a promoter of the present invention is an anti-sense RNA molecule for modulating the expression levels of an endogenous plant gene.
The invention further contemplates methods of demonstrating promoter activity for putative BRRV promoters. In this method, a putative BRRV promoter is operably linked to a reporter gene, for example a DNA encoding a green fluorescent protein or chloramphenicol acetyltransferase. A transgenic plant or plant cell is then generated by transforming the plant or plant cell with the resulting construct. The plant or plant cell, or a descendant thereof comprising the chimeric construct, is then assayed for the presence of the product of the reporter gene. In preferred embodiments, tissues from developing or mature plants are analyzed for the tissue-specific expression. "Tissue-specific expression" is expression of an RNA transcript or a protein product of translation of a transcript, that is at least five times greater than, more preferably at least ten times greater than, and more preferably at least twenty times greater in one tissue compared to another, when measured as a percentage of total RNA synthesis or total polypeptide synthesis in each tissue. One example of a promoter region identified from BRRV is an 821 bp sequence (SEQ ID NO: 9).
In some embodiments, the invention provides a plant or plant cell comprising a recombinant sequence derived from BRRV DNA. Preferably, the plant or plant cell comprises a recombinant sequence derived from BRRV DNA comprising a BRRV promoter sequence. More preferably, the plant or plant cell comprises a recombinant sequence comprising a BRRV promoter sequence operably linked to a nucleic acid encoding a polypeptide or an RNA molecule.
In some embodiments, the invention includes recombinant BRRV promoter constructs comprising multiple copies of a promoter sequence. Tandem repeats of a promoter G sequence or of any of the other promoter sequences can also be made and operably linked to a coding region for a desired expression product such as a reporter gene.
This example demonstrates isolation, characterization, and sequencing of BRRV DNA.
Blueberry red ringspot virions were isolated from blueberry leaf tissue using the technique of Gillett and Ramsdel (Gillett, J. M., and Ramsdell, D. C. (1984). Detecting the Inclusion forming Blueberry Red Ringspot Virus with ELISA. Phytopathology 74: 862). To obtain a sequence of BRRV, viral DNA, obtained from an infected blueberry plant in 1989, was digested with the restriction enzyme Eco RI. Separately, viral DNA obtained from infected blueberry plants at a later date was digested with restriction enzyme Xba I. Fragments digested with either enzyme were ligated into pUC 119 vector digested with either the Eco RI or the Xba I restriction enzyme. Recombinant plasmids were grown and maintained in E. coli DH5 alpha cells. Sequences of DNA fragments inserted into the pUC 119 vectors were determined using an ABI automated sequencer and "Big Dye" terminator technology. Once initial sequence was determined, unique primers were designed to "walk" through the sequence of the clone. Sequences obtained from the Xba I and Eco RI digests were then compared, and overlapping sequences were used to determine the relative positions of the fragments in an undigested virus.
This example demonstrates promoter activity in a recombinant fragment of BRRV sequence.
To demonstrate promoter activity in a fragment of BRRV sequence, an 821 bp fragment (SEQ ID NO: 9) was identified as containing a TATATAA box, at least one cis-acting transcription element, and a translation start sequence less than 100 base pairs from the TATATAA box. This fragment was operably linked to a cDNA encoding a green fluorescent protein (GFP). The resulting recombinant construct was used as a transgene to transform plants of the species Arabidopsis thaliana using standard procedures. Plant tissues comprising the transgene were assayed for fluorescence indicative of the presence of GFP. All tissues examined exhibited fluorescence, indicating that the sequence of the 821 bp fragment provided promoter activity that was able to direct the expression of the GFP cDNA
This example demonstrates promoter activity of putative BRRV promoters.
To investigate promoter activity in putative BRRV promoters, a GFP cDNA was operably linked to putative promoters of BRRV identified as above. For comparison, the GFP cDNA was also operably linked to a 35 S promoter of Cauliflower Mosaic Virus (CaMV). Arabidopsis thaliana plants were transformed, and transgenic plant tissues, in particular apical meristem, root, and leaf tissue were assayed for the presence of GFP. Relative brightness of fluorescence was estimated by eye using plants transformed with a construct comprising the CaMV 35 S promoter operably linked to GFP cDNA as a standard.
The lengths of putative promoter sequences tested for their ability to support expression of GFP cDNA in Arabidopsis thaliana tissue are as follows:
Promoter A--423 nucleotides
Promoter B--429 nucleotides
Promoter C--428 nucleotides
Promoter D--451 nucleotides
Promoter E--401 nucleotides
Promoter F--423 nucleotides
Promoter G--447 nucleotides
The results of testing the promoters are presented in Table 1. For promoter G, both truncated sequence fragments as well as tandem duplications of the sequence were also tested for their ability to direct GFP expression. Note that one putative promoter, Promoter A, provides the ability to direct tissue-specific expression of the transgene to the roots.
TABLE-US-00001 TABLE 1a Promoter: Apical Meristem Root Leaf BRRVG + N/Ab + BRRV G Short + + (+)c (221 bp) BRRV G Shortest + - - (219 bp) BRRV G tandem N/Ab + + repeat BRRV A - ++ - CaMV 35S +++ +++ + BRRV E - + + BRRV F - + + a"+++" indicates tissue strongly fluorescent for GFP. "++" indicates tissue exhibiting GFP fluorescence, but less intense than that of comparable tissue from plants transformed with the CaMV 35 S-GFP construct. "+" indicates tissue exhibiting GFP fluorescence, but less intense than that of comparable tissue exhibiting "++" intensity fluorescence. "(-)?" indicates extremely weak, possibly artificial, fluorescence. bN/A not attempted cFluorescence detectable but weak.
This example demonstrates discovery and demonstration of a minimal promoter derived from the BRRV DNA sequence.
To isolate a minimal promoter from BRRV, portions of the 821 base pair sequence described above were made. One such fragment, containing only 385 base pairs of DNA, was operably linked to the gene encoding green fluorescent protein, and the resulting construct was used to transform Arabidopsis thaliana plants as above. Leaf tissue from these plants exhibited fluorescence indicative of the presence of the green fluorescent protein, thereby demonstrating that a sequence no larger than 385 base pairs comprising promoter G exhibits minimal promoter activity, and can be used to direct expression of heterologous genes in transgenic organisms.
This example demonstrates the determination of a consensus sequence for a putative minimal promoter.
To determine a consensus sequence for a putative minimal promoter, a computer was used to align the sequences from each of the 7 putative promoters described above. Using standard sequence analysis algorithms, a consensus sequence was derived (SEQ ID NO: 10). This consensus sequence is notable for its high A-T content (approximately 90%). The aligned consensus sequence is shown in FIG. 2 in which consensus bases are shown in capital letters. Dashes represent more than one base as indicated in the possible bases at that position in the seven aligned sequences.
As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description be interpreted as illustrative and not in a limiting sense.
The inventor contemplates that many variations on the disclosed sequences, such as insertions, deletions, inversions, duplications, and substitutions are contemplated as within the scope of this invention. The inventor also contemplates that the promoters and their variants will exhibit distinct expression patterns in terms of both species specificity, tissue specificity and developmental specificity. The inventor also contemplates that sequences hybridizing to the blueberry red ringspot virus under stringent conditions, particularly the putative promoter sequences, will reveal useful activity, in particular promoter activity for use in directing expression of transgenes. It is contemplated that blueberry red ringspot virus nucleic acid sequences having lengths of about 10, 15, 20, 25, or 50 base pairs can be used for hybridization under stringent conditions to isolate new examples of useful sequences having homology to the virus. Sources of DNA for hybridization can be other viruses, plants, animals, fungi, and prokaryotes.
All references cited in this specification are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art relevant to patentability. Applicant reserves the right to challenge the accuracy and pertinency of the cited references. As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description be interpreted as illustrative and not in a limiting sense.
1018241DNABlueberry Red Ringspot Virusmisc_feature(7888)..(7888)unknown nucleotide 1ggtatcagag cttgagctac aaatagttca aactcgaaat atcttaatgg aaattcccga 60tttatcacaa ttatcaaata tagtactcac tgataatcca ggaaccaatc caggaatccc 120gtataaagga aagattataa gtatacctaa gaatttacta ccagagaaaa ctgagtattc 180attagcctat gatggatctc atgaacaacg aatattacct atgttaagat ttattactgc 240tatattaaat aatcaaaccg aaatattaag tttcctatct attaatcaaa taaagtcccg 300gacagtattc acagaaaaat actttgaaga acaaaagaat aatatatcta tacagcttga 360acaaaatcgt aaaaatatat tccaaaagtt agaagagcta aaaggattat atgataaaaa 420acaagatact attatacaac actctaatag gataataaac ctgataaccg aaatagaaca 480taaaaaagac ttagacgaaa taaaaaatac gctaaaagaa atcaaaaagg atttagtcga 540aaataataac caaaaagaag ttaaagaatt aatcgaaagt ctaaaagata taagtgatcg 600aatataaatg actgaaggta acggaaacgg aaaattaacc gaaaaaatag aaaatatgat 660attaaaatat gaagagttag aaaaagtagt aaaacaagta agtcacgaaa tacatgaaat 720aaaagaacaa gtagaagtca aaacgacata aaagaattca aggaatcaaa atactataac 780aacactatcc gagggaatgc ccaacctaca aaagaaatga tcatcgataa tcaaccagaa 840ccatgttatc aaaaagattc accttttcca catctatatc atacctttga tccacaatta 900aataatataa cagatatgct tatgtatata ataaaaaatt gctgttgcaa agacaagcat 960gaacagaaag aaaaacaacc tataatacat cctaaaatat tagaagaaaa agaacgacaa 1020ataaaagatt tagaagatca cataaaagag ttagaagaac acagatgcat aactatttta 1080gatttaaaga aatattttgg aaataatttt agaggaaaag aggaaagaag accaatagaa 1140tatctagaag accaacctag ccctagttat agaggaagaa aaccattatg gcctcagata 1200atatagatca gcaaatcgaa gaaattagaa atatcatgaa taatcttaaa atagaaaaga 1260ctgaagaaaa tgaaataata tttggaaatg aatcagaaaa tagtgattat gatgtaagaa 1320tggatgatgt aaaagaagaa ccaggaacta gtaatttcga agaaagatgg aagagaaaaa 1380gaaatccaac ttacgaatat gaaccatatc ataccgaccc atttattaat gcagaagatt 1440atggatataa aagaggattc tataaaaata gacaaggtaa atggaaaaaa cctaaagaac 1500caggtccagt aacaggacga ttagaagatg gaatattaaa tctagattgt ttaactaatg 1560gagaagaatt attaaaacaa tggacagcaa aacaatcatt atcaacacaa attgatgcta 1620ccatccgaga catggatgct gagaattata acaaatattt aatatataaa acatcaggtg 1680tagtatttaa ttatatagtt gatatagaca tacctaatat aagctctatg aaaaacctag 1740aaatattaga ggaaatagct accaaaatat atcaagaatt cctaggagga atgagtaccc 1800aaagagccac agcggcagat aaacatgatc aagaaagaat aaaatacata ttacataaaa 1860tgaaaatatg tgacatgtgc gaattcgaag aattctactg tcaatttata cattattatt 1920atatgttaga atctaaagag agagctgaat atatgaatgt atttatccag aaactaccag 1980aaccccaatt aatacccgac acgttacgag gtatagccaa agtaataaga gcatacatat 2040tattacaatg tctaaggaac aagaaaaaat gcaattaatt aatgtaacaa atgttgccca 2100aaattcgaat atataccaca taaatttggt tgttcaccaa attcttcttt tcggagggga 2160agaaagcgaa ctaaaccaaa tattcaaaat ataaaaacgc aaatatcata cctttaaacc 2220atggtataaa aagaaacgat atcgtatgta taaaagaaaa tatcaaccca aatataaaca 2280aagatattgg aaaaataaaa gtaatcagaa atattgccct aaaggaaaaa aggattgcaa 2340gagttggatc tgtcaagaag atggacacta tgcaaatgaa tgccctaaca aagataaaag 2400aagagataaa gtaaagctac tagaacaatt atcacaagta aatctcgaac caatagaaaa 2460tgataatata tcagaagaag aattatggta tttacaaact gatgaagaat cagaagaaga 2520aaatagttca gatgaatcag aacaatactt ttatcaagat aacaatcagt cagaagacga 2580tattagcata tattgatcag ggtgcatcat tgtccctata accagaacat aatttaccaa 2640aacaattatg gaaagaaaat aagaacccaa ttacaataag agtagccgat aagagagact 2700acaattaata aagtagccct tatgattaca atattaatag aaaaaaggaa attccttgta 2760cccactatat atcaatttga ttcaggagta ccaatgatta taggaaataa ctttttaaga 2820ttatattacc cattttgtca atatctatct tatataacat taagatgtcc taaaatgatc 2880aatcaaaaac aagaagtgat taaaataccg atacatcaca gttcacaatt aataaaagca 2940aagttactaa acttagtaac aaatattgaa gaacaattat taatggaaca agttaataaa 3000atattacaag aaagattctc actagatttg cttggagaaa agaataaaaa taaagaacta 3060atagaaataa aattaaaaga cccaaatgct gaaatatttg taccaaataa tataccttat 3120acccaaagag atatagaaga attcaaagaa gatatggaag atctaataaa caaaggatta 3180ataagaccaa gtgaaagtcc acatagtgcg ccagcattct atgtcgaaaa tcattcagaa 3240ataaaaagag caaacagaag aattgtaata aattataaag ctatgaatga agccaccatt 3300ggaacaccta aaacattacc cagagcagat ttataatgaa cagattagaa cagcacaaag 3360gtaaaatttg gttctcaaca ctagatgtaa aatcagccta ctggcaatta agattaactg 3420aagaatctaa accattaaca gcatttagtt atccacctca aaaacattac gaatggaatg 3480tgttacctat gggattaaag caagctccag gtatttttca agaatttatg aatagaagtt 3540tacataattt ataacatata tgtttagtat atgttgatga tattattata ttttcagaaa 3600aggataaaaa tgatcattta tcaaaagtat tacaagtttt aaaacgatgc gaagatgaag 3660gaatcatctt atcacagcca aaagcaaaaa tagcacataa agaaaatgca cataaacaat 3720tgatttcttt ggattacata tttcagaagg agaaataata ctacaaccac atattttgga 3780aaaattagta ttattccctg atgaaattca gaatcgaaag caattacaaa gatttcttgg 3840aaatttaaat tatataagtg aaaaaggatt ttttaaggat tttgcaaaat atcgaaaaga 3900tttacaaaag aaagtatcag aaaaagtgcc atggaaatgg acttcctatg acactagtca 3960agtgcaagca ctcaaggctt taagccaacg attaccaaga ttatacaatg caaaagaatc 4020agacttgctg ataatcgcta ccgatgctag taatggccat tggggagcag ttatgacagc 4080agtcactcca gtacatatca ggaattatgg aatatccctt gaggatttat tcccaaaaga 4140tcagcataca gcacaagcat tatcatctca atatcagttc ttcggaacaa aggactttgt 4200ccaaaaagaa ttacttacta aatatgctag tggaacattc acggataccg aaaaaagata 4260tcctatccat gaattggaaa cattagcagt attacaaact tttcgaaaat ggaaagttga 4320tttactatca aaacctttta ttttaaaaac agattcaaaa tatgttacag gatttttgag 4380atataaaata aaagccaatt ataatcaagg acggttgatt agatggcaac ttgaattatc 4440acaattcaac tataaaactt tttatataaa aggatcagaa aattatggcc ctgacaccct 4500aaccagggaa tggaaggagc tataaagaca ttggagaatc aaatcagcac taaagagcaa 4560tcatttcaag caaaaaagca gaagttagca gaactcgaag cagagatcaa caacctcaga 4620tcaacattag ccatactcag tggagatagc agcaaactat caccatcagc cccagaaaca 4680acaaagcatt cagtattagc caatattgaa gaagtcaaca aacaagtggc agcagccaga 4740atcaagaagg aatattatgt catttttaat ggacccatga aaggaatcta tgacgaatgg 4800cataaagcag caccacacat tcaaggacaa tccagcatca ttcacaagaa atatccaacc 4860attgatgaag caaaaaaggc tcttggagga agctacgcag caatcaccaa cgcaccagca 4920tcaccaaagg atacaaaagt actattggga agattcaaag tccctttagc accaacgatt 4980gattcaattc aaactattga gtcaaaaatg aaagcattaa aagttactca gaagaaatat 5040aatgattata tggaaatcgt acacaattac aaggatcaac ataaatcatc acatttctat 5100ccgaagtacc gagatacaat tggatataaa gcaataatct tgaccgaagc atcagcactc 5160cccacctatg aattgttcaa aaatggatta gccgacacta tctatttttc ggacataaaa 5220ccattcaatg attttcctga acgaatcaag caaaccatca acaattattt caagaggttc 5280gccaaggaaa gaccatgcta catcaaatta ttcagcactc atccaacttt cagtatacaa 5340ggagaagaag acatgcccag ctatgcagta ctacagatcg gaattagcaa tggagatatg 5400cctttaatgg acacccttca tatgccagta cccaagcatg aggagttaaa acaaatcaat 5460ttacagaatt tcattggaat tattaatcat ttatccaatt taacagcaaa cattaaaatg 5520ctgtataaat ccgatacaat gattatctat tccaaagcca acaaggagat agaaccagat 5580caagaagaag tattcattca atttgaaaag aattttattg aaaataaaat tccaaaaatg 5640gcgggggaga tgaagaaaga attatgcaat catatgacca aagaagatca tccaggacat 5700tattgtcaat attgtccatt cattcagcag gatgataagc agtcagcatc aagtgaagaa 5760gaaaagatca ccatggaagt ggaggaataa aacgtcttca tccatcacta tcaagatgta 5820gacattacaa tgtaaagcta cggctattat taggcttaga atctacgcca atgtaaagta 5880gattccctta tcttttattt tgtaagtttg aagtccggat ctgagttacg cctgtgagaa 5940ttcctgtata taaggacgac ctatcctatg tttgtagtca gagtgttttc catatgtcga 6000ataaacgtct aacgttttga aggtgtcgcc ctttcactct ccaatgttga gttcttcgta 6060tgaagatatc tgaagagcac atcctatccc tatataccct atccttaacc tatcccaaat 6120tctatcccaa aggttttgtt gcagaagcaa aactttcatg gattcaagac aggattcctc 6180gaagaacaag tatatgccta aactaccact attttatctc tatctgtgtg tacttattca 6240tttcattata tcgattttgt tattactctt tcttgttatc atgatatgtc aaatcaaact 6300actttgttga acaattatta tctacagtct tatattattg gaattataaa tgaaagaata 6360agattaatat tagattgaca caggagcagc tatgtccttt ataaaacaat gatctcgttc 6420atcaataatc attaacacct attcaacctt atacagtaaa aggatattta gatcatcata 6480caattggaga caaatactta gtagaacatt ctaccaaaca taaatgtcat attaagtaac 6540acctttaaca aaacattatt actaaaaaca actcaaacaa aagcagatgt attaggagcc 6600gaagtattat tcggaatgga ttttttagat agctttgaat catattcaat cacaaaggaa 6660caactaatac taagagaagg aaatattact cattatatac caagaatcac agtcgataca 6720gacaccataa gagaaaccct aaactattaa agatgtcatc acctattata ttagaccaag 6780caggagtagt ccaaaatcaa gaacagaatc aaatatcaaa agttagtaat caaaatcaga 6840tgtcaagaat agaagatcaa aaccaatctg aagaaatcag acatatcaat caaatctttg 6900atattataca atcaaatgaa acaaatgatt attatggagt cgatgtccaa ctagaaagat 6960ctaaactaaa acaaatagca aaagaaggaa aattaacatt aaaatcagga caaggaataa 7020aatcagaagg attcttacca tctattacta gaaagaatat attatatcta ggaaaattta 7080caagcgaaca accattagaa ataagcacag cagtaggaca agaagcacta tcattagtta 7140atggaaagca gatagcagac cgaatagcta aaatgaaaca atcagataaa gaaaaaattc 7200agtatataca tataagcaca atacaaatac tagttaagtc tacttatgcc tcaatagata 7260cacccatgga tattatagtt attgataata gaataatttc taaaaataag aaagaacaag 7320ttctaggaat aattaaagga aatctgaaat atggagttat taagtttgat gtaagtttac 7380acttcgctat accattagta actaagaact taagtcaatc aataggaata ttatataagt 7440tccacagaca ggatctgatg gaaaaaggag attacccact tagtattacc tactctgtag 7500gatatgcatt aagcaatagc caccatagtg ttgattatat agatcaagag attatacata 7560tagacgattt atttaaaaat actagtacta aattagtaac atttgagaaa aagaatgaaa 7620acgccaacga catatttaga gcaccaccag taagaatgat taaaccaaga gaggatttat 7680caaaaccaac tataacagat gtcacagacc cattaaaacc aaccaccagt agttctatcc 7740aattagcacc tccacctaac ctacatagaa aaccagagag catgcagaac ttagaaaagc 7800aaatacagga attaagaaga acagttacaa acctcaatga caaaatatga gctatcagta 7860tagaagagga ccatatatga aaggaagnta taaacgatgt ctcatagaag atttagaacc 7920catgaaggac atactagtaa ctactaagag agaaatatta gaacaatttt ccagatctga 7980agaaagatca aaaataatta gcaatttgca aggattaatt gattatttag tcaaaagaca 8040gaaacgagaa gcagaaaatc cggaattaac catccaagaa aagatattaa cgagattgaa 8100cagcattgaa gaaaaattag agaagtctaa ttcattttca tcaatatttg atgatctaga 8160agaaacttct caccaaacaa atcaggtcaa tattccgcca tctagtgaaa actagtaagt 8220tgaccgcccc ggtccgtttt t 82412423DNABlueberry Red Ringspot Virus 2gttaaagaat taatcgaaag tctaaaagat ataagtgatc gaatataaat gaccgaaggt 60accggaaatg gaaaattaac cgaaaaaata gaaaatatga tattaaaata tgaagagtta 120gaaaaagtag taaaacaagt aagtcacgaa atacatgaaa taaaagaaca agtagaagtc 180gaaagcgaca taaaagaatt taaggaatca aaatactata acaacactat ccgagggaat 240gcccaaccta caaaagaaat gatcatcgat aatcaaccag aaccatgtta tcaaaaagat 300tcaccttttc cacatctata tcataccttt gatccacaat taaataatat aacagatatg 360cttatgtata taataaaaaa ttgctgttgc aaagacaagc atgaacagaa agaaaaacaa 420cct 4233429DNABlueberry Red Ringspot Virus 3tgtaaaagaa gaaccaggaa ctagtaattt cgaagaaaga tggaagagat aaagcaatcc 60aacttatgaa tatgaaccat atcataccga cccatttatt aatgcagaag attatggata 120taaaggagga ttctataaaa atagacaagg taaatggaaa aaacctaaag aaccaggtcc 180agtaacagga cgattagaag atggaatatt aaatctagat tgtttaacta atggagaaga 240attattaaaa caatggacag caaaacaatc attatcaaca caaattgatg ctaccatccg 300agacatggat gctgagaatt ataacaagta tttaatatat aaaacatcag gtgtagtatt 360taattatata gttgatatag acatacctaa tataagctct atgaaaaacc tagaaatatt 420agaggaaat 4294428DNABlueberry Red Ringspot Virus 4tatcagaaga agaattatgg tatttacaaa ctgatgaaga atcagaagaa gaaaatagtt 60cagatgaatc agaacaatac ttttatcaag ataacaatca gtcagaagac gatattagca 120tatattgatc agggtgcatc attgtcccta taaccagaac ataatttacc aaaacaatta 180tggaaagaaa ataagaaccc aattacaata agagtagccg ataagagaga ctacaattaa 240taaagtagcc cttatgatta caatattaat agaaaaaagg aaattccttg tacccactat 300atatcaattt gattcaggag taccaatgat tataggaaat aactttttaa gattatatta 360cccattttgt caatatctat cttatataac attaagatgt cctaaaatga tcaatcaaaa 420acaagaag 4285451DNABlueberry Red Ringspot Virus 5ttaaaatacc aatacatcac agttcacaat taataaaagc aaagttacta aacttagtaa 60caaatattga agaacaatta ttaatggaac aagttaataa aatattacaa gaaagattct 120cactagattt gctaggagaa aagaataaaa ataaagaact aatagaaata aaaattaaaa 180gacccaaatg ctgaaatatt tgtaccaaat aatatacctt atacccaaag agatatagaa 240aaagtccaca tagtgcgcca gcattctatg tagaaaatca ttcagaaata aaaagagcat 300taagaagaat tgttataaat tataaagcta ttgaatgaag ccaccattgg aacacctaaa 360cattacccag agcagattat ataatgaaca gattaaaagg aaaaatatgg ttttcaacac 420tagatgtaaa atcagcctac tggcaattaa g 4516401DNABlueberry Red Ringspot Virus 6tttttcaaga atttatgaat agaagtttac ataatttaga acatatatgt ttagtatatg 60ttgatgatat tattatattt tcagaaaagg ataaaaatga tcatttatca aaagtattac 120aagttttaaa acgatgcgaa gatgaaggaa tcatcttatc acagccaaaa gcaaaaatag 180cacataaaga aattgatttc tttggattca tatttcagaa ggagaaataa tactacaacc 240acatattttt ggaaaaatta gtattattcc ctgatgaatt cagaatcgaa agcaattaca 300aagatttctt ggaaatttaa attatataag tgaaaaagga ttttttaagg attttgcaaa 360atatcgaaaa gatttacaaa agaaagtatc agaaaaagtg c 4017423DNABlueberry Red Ringspot Virus 7cccttgagga tttattccca aaagatcagc atacagcaca agcattatca tctcaatatc 60agttcttcgg aacaaaggac tttgtccaaa aagaattact tactaaatat gctagtggaa 120cattcacgga taccgaaaaa agatatccta tccatgaatt ggaaacatta gcagtattac 180aaacttttcg aaaatggaaa gttgatttac tatcaaaacc ttttatttta aaaacagatt 240caaaatatgt tacaggattt ttgagatata aaataaaagc caattataat caaggacggt 300tgactagatg gcaacttgaa ttatcacaat tcaactatag aactttttat ataaaaggat 360cagaaaatta tggccctgac accctaacca gggaatggaa ggagctataa agacattgga 420gaa 4238447DNABlueberry Red Ringspot Virus 8tttttaccat ctattactag aaagaatata ttatatttag gaaagttcac cagcgagcaa 60ccattagaaa ttagcacagc agtaggacaa gaagccttat cattagttaa tggaaagcaa 120atagcagaca gaataacaaa aatgaaacaa tcagataaag aaaagattca gtatatacac 180ttaagcacaa tacagatatt agttaaatcc acctatgcct caatagatac accaatggat 240attatagttg ttgataatag aataatttct aaaaataaga aagaacaagt tctaggaata 300attaaaggaa atctgaaata tggagtatta agttcgacgt aagtttacac ttcgccatac 360cattagtaac taagaattta agccaatcta taggaatatt atataaattc cacagacaag 420atctgatgga aaaaggagat tacccac 4479821DNABlueberry Red Ringspot Virus 9ataccaagaa tcacggtaga tacagaaact ataagagaaa ctttgaatta ttaagatgtc 60atcacctatc atattagatc aagcaggagt aatccaaaat caagaacaaa atcaagaaca 120gaatcaaatg tcaagagtca ataatcagaa tcagatgtca aaaatagaag atcagaatca 180atcagaagaa atcagacata ttaatcaaat ctttgatatt atccaatcaa atgaaacaaa 240tgattattat ggagtcgatg tccaactaga aagatctaaa ctaaaacaaa tagcaaagaa 300ggaaaattaa cattaaaatc tggacaagga ataaaatcag aaggattttt accatctatt 360actagaaaga atatattata tttaggaaag ttcaccagcg agcaaccatt agaaattagc 420acagcagtag gacaagaagc cttatcatta gttaatggaa agcaaatagc agacagaata 480acaaaaatga aacaatcaga taaagaaaag attcagtata tacacttaag cacaatacag 540atattagtta aatccaccta tgcctcaata gatacaccaa tggatattat agttgttgat 600aatagaataa tttctaaaaa taagaaagaa caagttctag gaataattaa aggaaatctg 660aaatatggag tattaagttc gacgtaagtt tacacttcgc cataccatta gtaactaaga 720atttaagcca atctatagga atattatata aattccacag acaagatctg atggaaaaag 780gagattaccc acttagtatc acctactcag taggatatgc a 82110151DNABlueberry Red Ringspot Virusmisc_feature(6)..(6)a or g or c or t, unknown, or other 10anacantatc caatangttt acaagantan aaaatanaan atcattgata anataaaaga 60gatataanat taaaacaatt aanattaaaa ncatntgaat taagccnnaa atntaataac 120aaanaattta tnaaanttna tnntatataa a 151
Patent applications by Board of Trustees of Michigan State of University